CN109355217B - Bacillus subtilis and microorganism formulation and its application - Google Patents
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Abstract
The present invention relates to microbial pesticide technical fields, disclose the application in a kind of bacillus subtilis (Bacillus subtilis) and microorganism formulation and its application, the especially soft rot caused by anti-banana Erwinia chrysanthemi (Erwiniachrysanthemi).The deposit number of the bacillus subtilis is CGMCC No.15506, and its prevention and treatment that can be exclusively used in squirter caused by Erwinia chrysanthemi.There is significant in vitro and living body preventive effect to squirter using bacillus subtilis made from this method, reachable 25.43mm average to the antibacterial circle diameter of Erwinia chrysanthemi, can reach 97.73% to the average control efficiency of squirter caused by Erwinia chrysanthemi.
Description
Technical field
The present invention relates to microbial pesticide technical fields, and in particular, to a kind of bacillus subtilis (Bacillus
Subtilis), microorganism formulation and its anti-squirter and inhibit Erwinia chrysanthemi (Erwiniachrysanthemi) in
Application.
Background technique
Erwinia category (Erwinia) bacterium is a kind of main plant pathogen for causing plant soft corruption, the European bacillus of chrysanthemum
It is one of.
At abroad, banana bacterial soft rot occurs in western Bangladesh for the first time in nineteen eighty-three.Then, in the perfume (or spice) on the ground such as India
The successive occurrence injury of Jiao Yuan.The disease is found for the first time in Guangdong Fanyu banana plantation within 2009.2012 in the double versions in Yunnan Province west
State banana plantation of receiving finds squirter, and pathogen is Erwinia chrysanthemi.
Erwinia chrysanthemi main harm banana caulo and growing point cause leaf sheath, false stem in soft rotten shape, yellow leaf, sternly
The rotten lodging of plant vacation stem rot of weight, and bad smell is given out, cross-sectional disease plant vacation stem, the disease ecto-entad expands in soft rotten shape
Exhibition sprawling, soft corruption position is in dark brown, is fallen ill with 8-9 month high-temperature high humidity season heavier, equal in banana Adult plant and Seedling Stage
There is generation, when serious, diseased plant rate reaches 20%-30%, and has the tendency that aggravating year by year, has seriously affected the yield and product of banana
Matter.
Prevention and treatment for squirter caused by Erwinia chrysanthemi, current main measure have select anti-disease tolerant variety,
The methods of efficent rotation and chemical prevention, chemical agent are mainly antibiotics medicament such as agricultural streptomycin, spring flower bud mycin etc., but
It is limited to the generation and sprawling of a small range control disease, when banana plant is larger, also to increase formulation rate, environment is caused
Pollution and raising cost.As pathogen drug resistance generates, biological control is because its environmental pollution is small and its can play lasting control
Disease effect and generally by the attention of researcher.Currently, there is no squirter microorganism formulation caused by anti-Erwinia chrysanthemi
The relevant report of Field information.Therefore, carry out the research and development tool of squirter biological agent caused by Erwinia chrysanthemi
There is highly important realistic meaning.
Summary of the invention
Draw the object of the present invention is to provide a kind of bacillus subtilis strain, microorganism formulation and its in anti-Erwinia chrysanthemi
The application in squirter risen, the anti-banana bacillus subtilis microbial preparation containing the bacterial strain draw Erwinia chrysanthemi
The squirter risen has significant in vitro and living body preventive effect.
To achieve the goals above, first aspect present invention provides a kind of bacillus subtilis, the bacillus subtilis
Deposit number is CGMCC No.15506.
Second aspect of the present invention provides a kind of microorganism formulation, and the active constituent of the microorganism formulation is above-mentioned withered grass gemma
The metabolite of bacillus.
Third aspect present invention provides a kind of preparation method of microorganism formulation comprising following steps:
(1) above-mentioned bacillus subtilis carries out solid culture in solid medium and obtains test tube species；
(2) liquid seed culture medium is prepared, and is inoculated with test tube species in the liquid seed culture medium and carries out Liquid Culture
Liquid seeds are made；
(3) liquid fermentation medium is prepared, and is inoculated with liquid seeds in the liquid fermentation medium and ferments.
Fourth aspect present invention provides one kind above-mentioned bacillus subtilis or microorganism formulation in anti-squirter
In application.
Fifth aspect present invention provides above-mentioned bacillus subtilis or microorganism formulation and is inhibiting Erwinia chrysanthemi
In application.
Bacillus subtilis and microorganism formulation of the invention has significant in vitro and living body anti-squirter
Effect, it is average to the antibacterial circle diameter of Erwinia chrysanthemi up to 25.43mm, it is averaged to squirter caused by Erwinia chrysanthemi
Control efficiency reaches 97.73%.
Other features and advantages of the present invention will the following detailed description will be given in the detailed implementation section.
Biological deposits
Bacillus subtilis (Bacillus subtilis) of the invention, this paper abbreviation XJ130301, in March, 2018
It is deposited within 26th China Committee for Culture Collection of Microorganisms's common micro-organisms center (referred to as: CGMCC), deposit number is
CGMCC No.15506, the address of the collection are as follows: city, BeiJing, China, North Star West Road 1, Chaoyang District institute 3, the Chinese Academy of Sciences
Institute of microbiology, postcode: 100101.
Specific embodiment
Detailed description of the preferred embodiments below.It should be understood that described herein specific
Embodiment is merely to illustrate and explain the present invention, and is not intended to restrict the invention.
First aspect present invention provides a bacillus subtilis, and the deposit number of the bacillus subtilis is CGMCC
No.15506.Bacillus subtilis of the invention is from one banana of the Chinese Xishuangbanna Prefecture, Yunnan Province Jinghong City town Meng Han Olive Dam
It is isolated on one plant of banana, the 16s RNA sequence phase of the bacterial strain and bacillus subtilis TOA (accession number: CP005997.1)
It is 99% like property.
The morphological feature of above-mentioned bacillus subtilis CGMCC No.15506 are as follows: formed on NA culture medium flat plate dirty white
Color or yellowish bacterium colony, bacterium colony rough surface is opaque, and wrinkle mould is commonly formed when growing in liquid medium.Gram sun
Property bacterium, aerobic bacteria.Individual cells (0.7-0.8) × (2-3) micron, peritrichous, no pod membrane can move；Gemma (0.6-0.9)
× (1.0-1.5) micron, ellipse arrive column, and central or slightly inclined positioned at thallus, thallus does not expand after sporulation.
NA culture medium: glucose 10g, agar 20g, beef extract 3g, yeast extract 1g, peptone 10g, distilled water
1000mL, pH=7.0.28-30 DEG C of cultivation temperature all may be used.
Second aspect of the present invention provides a kind of microorganism formulation, and the active constituent of the microorganism formulation is above-mentioned withered grass bud
The metabolite of spore bacillus.
Third aspect present invention provides a kind of preparation method of microorganism formulation, includes the following steps:
(1) above-mentioned bacillus subtilis carries out solid culture in solid medium and obtains test tube species；
(2) liquid seed culture medium is prepared, and is inoculated with test tube species in the liquid seed culture medium and carries out Liquid Culture
Liquid seeds are made；
(3) liquid fermentation medium is prepared, and is inoculated with liquid seeds in the liquid fermentation medium and ferments.
Preferably, in step (1), the bacillus subtilis is inoculated with by the way of inclined plane inoculating, the solid
Culture is 25-30 DEG C of culture 40-50h in temperature；The solid medium includes: glucose 10-12g/L, agar 15-20g/L,
Beef extract 3-5g/L, yeast extract 1-3g/L, peptone 10-15g/L, pH=6.5-7.5.
Preferably, in step (2), test tube species are inoculated into liquid seed culture medium, and the Liquid Culture is 25- in temperature
30 DEG C, revolving speed be 200-250r/min under conditions of handle 45-50h；The liquid seed culture medium includes: peptone 10-
15g/L, beef extract 3-5g/L, sodium chloride 10-12g/L, pH=6.5-7.5.
In the step, liquid seed culture medium preferably sterilizes at 120-125 DEG C 20-30min, it is cooling after in 100mL liquid
0.5-1.5cm is accessed in body seed culture medium2Test tube species, and at 28-30 DEG C revolving speed be 220-250r/min shaking table
Upper culture 45-50h obtains liquid seeds.
Preferably, in step (3), liquid seeds are connecing for 0.05-0.1:1 according to volume ratio in liquid fermentation medium
Kind amount inoculation, and 45-50h is handled under conditions of temperature is 25-30 DEG C, revolving speed is 200-250r/min；The liquid fermentation
Culture medium includes: sucrose 20-23g/L, peptone 10-15g/L, yeast extract 5-8g/L, potassium dihydrogen phosphate 3-5g/L, ammonium sulfate
5-8g/L, calcium carbonate 2-4g/L, pH=6.5-7.5.
In the step, liquid fermentation medium preferably in 120-125 DEG C of sterilizing 20-30min, is inoculated with liquid strain after cooling
Son, and fermentation liquid is made in culture 45-50h on the shaking table that revolving speed is 220-250r/min preferably at 28-30 DEG C.
Further, the method can also include that the fermentation liquid obtained to fermentation purifies；The purification side of fermentation liquid
Method includes: that fermentation liquid is extracted using organic solvent, removes organic solvent later.The organic solvent is preferably methanol, ethyl alcohol, third
At least one of ketone, methylene chloride and ethyl acetate.
Wherein, when addition organic solvent is extracted in fermentation liquid, it is preferred to use centrifugation promotes separation, convenient for removal
Other impurities in organic solvent and fermentation liquid.
Preferably, acetone is added in fermentation liquid to be centrifuged, after removing acetone, then is diluted with water to original fermentation liquor body
Product, can be prepared by microorganism formulation.
Fourth aspect present invention provides one kind above-mentioned bacillus subtilis or microorganism formulation in anti-squirter
In application.The pathogenic bacteria of the squirter are preferably Erwinia chrysanthemi.
Fifth aspect present invention provides one kind above-mentioned bacillus subtilis or microorganism formulation and is inhibiting chrysanthemum Euclidean bar
Application in bacterium.
Microorganism formulation in the present invention containing bacillus subtilis strain to squirter have it is significant in vitro and
Living body preventive effect prevents and treats squirter significant effect caused by Erwinia chrysanthemi, can be used for the prevention and treatment of soft rot in banana crops.
Bacillus subtilis of the invention and the microorganism formulation with anti-Erwinia chrysanthemi are right in inhibition zone method test
The antibacterial circle diameter average out to 25.43mm of Erwinia chrysanthemi mycelia growth, the results showed that bacillus subtilis and tool of the invention
There is the microorganism formulation of anti-Erwinia chrysanthemi that there is very strong inhibitory activity effect to Erwinia chrysanthemi growth.
The soft rot of bacillus subtilis of the invention and the microorganism formulation with anti-Erwinia chrysanthemi to potting banana
Significant control efficiency is shown, average control efficiency reaches 97.73%.
The present invention has many advantages, such as that low cost of material, simple production process, control efficiency are good, before having preferable application
Scape.
The present invention will be described in detail by way of examples below.
Embodiment 1
Microorganism formulation is prepared in accordance with the following steps:
(1) test tube species culture
Bacillus subtilis strain XJ130301 is inoculated into test tube culture medium slant, cultivates 48h at 28 DEG C,
Obtain test tube species.The formula of test tube solid medium are as follows: glucose 10g, agar about 18g, beef extract 3g, yeast extract 1g,
Peptone 10g, distilled water 1000mL, pH=7.
(2) liquid seeds culture
Using triangular flask shaking flask culture, the liquid seed culture medium described in loading 100ml in each triangular flask, 121 DEG C
Sterilize 20min, accesses 1cm after cooling2Test tube species, at 28 DEG C in revolving speed be 220rmin-1Shaking table on culture 48h obtain liquid
Body seed.The formula of liquid seed culture medium are as follows: peptone 10g, beef extract 3g, sodium chloride 10g, distilled water 1000ml, pH
=7.
(3) preparation has the microorganism formulation of anti-Erwinia chrysanthemi
Using triangular flask shaking flask culture, 200ml liquid fermentation medium, 121 DEG C of sterilizings are packed into each triangular flask
20min, it is cooling after access 10ml step (2) cultivate obtained liquid seeds, at 28 DEG C in revolving speed be 220rmin-1Shaking table
Upper culture 48h obtains fermentation liquid.Liquid fermentation medium formula is: sucrose 20g, peptone 10g, yeast extract 5g, biphosphate
Potassium 3g, ammonium sulfate 5g, calcium carbonate 2g, distilled water 1000ml, pH=7.
100 isometric weight % acetone are added in fermentation liquid, are 4000r with revolving speed after shaking table vibrates 12h
min-1Centrifuge be centrifuged 15min, take supernatant and on a rotary evaporator 25 DEG C of evaporative removal acetone, be diluted with water to primary
Zymotic fluid volume is to get the microorganism formulation for arriving anti-Erwinia chrysanthemi.
Embodiment 2
Antibacterial Activity test:
Antibacterial Activity is carried out using filter paper measuring method.By banana Erwinia chrysanthemi, (number is
33242) bacteria suspension 0.5ml, being inoculated in NA plate using rubbing method, (preparation method is: after taking distilled water 1000mL to boil, adding
Enter glucose 10g, agar 20g, beef extract 3g, yeast extract 1g, peptone 10g, pH=7.) on, by the filter paper of sterilizing
(5mm) is immersed in microorganism formulation made from embodiment 1, fully absorbs it, after drying in the shade, is taken and was impregnated with aseptic nipper
The filter paper to be measured of test solution is lain against in the culture dish containing test bacterium, three filter papers of every ware, 3 repetitions, is sky with clear water
White control.Culture dish after preparing is placed in 30 DEG C of constant incubator cultures, observes after 48h, is measured and is pressed down with crossing method
Bacterium loop diameter.
By test, 5 repetition antibacterial circle diameter measurement results be respectively 24.45mm, 25.58mm, 25.36mm,
26.12mm, 25.63mm, average out to 25.43mm.The results showed that containing bacillus subtilis XJ130301 of the invention
Microorganism formulation has stronger inhibiting effect to the growth of Erwinia chrysanthemi.
Embodiment 3
Greenhouse pot culture live test:
The test preparation method of banana seedlings: it in vinyl house, after the banana seedlings of tissue culture clean root, transplants in nursery
In bag.It after banana seedlings grow 3-4 piece leaf (about 1 month), then transplants in the seedling-growing container of diameter 30cm, through normal water drenching after transplanting
Moisturizing, weekly fertilising (every plant 2g/ times, compound fertilizer's application soluble in water, based on mass fraction, the N-P in compound fertilizer2O5-K2O is
15-15-15) 1-2 times.It is spare after banana seedlings grow 5-6 piece leaf (about 1 month).
Microorganism formulation prepared by embodiment 1 with anti-banana Erwinia chrysanthemi pours to be filled in growing way consistent above-mentioned
On potting banana plant root, after 7d, the banana seedlings of potting are carried out to hurt root from 2 different directions with the scissors of sterilizing, so
By the banana Erwinia chrysanthemi for 24 hours of 30 DEG C of constant temperature incubations on NA plating medium, (number is afterwards33242) with sterile
Water is made into 1 × 108The bacterium suspension pouring banana seedlings of cfu/mL hurt root position, every plant of 40ml, each 10 plants of processing, 4 weights
Multiple, control group does not pour the microorganism formulation with anti-banana Erwinia chrysanthemi.
Observation has the microorganism formulation of anti-banana Erwinia chrysanthemi soft to banana caused by banana Erwinia chrysanthemi after 14d
The inhibition situation of maize ear rot morbidity, counts each processing disease index, and calculate control efficiency.
Investigate grade scale:
0 grade: plant is disease-free without withered and yellow symptom complete stool；
1 grade: plant footing leaf and leaf sheath occur withered and yellow symptom scab number 2 or less；
3 grades: yellow leaf leaf sheath necrosis scab number 3-5 following in the middle part of plant；
5 grades: plant middle and upper part yellow leaf leaf sheath necrosis scab number 6-8；
7 grades: whole strain jaundice leaf sheath necrosis scab number 9 of plant or more；
9 grades: the whole strain of plant is withered, lodging scab gathers, and blade is seriously withered.
The calculating of drug effect uses following two formula:
Disease index=[(∑ (the diseased plant number disease grade numbers of sheets at different levels × opposite value of series represents series)/(investigation total strain number leaf
Number sum × highest value of series highest represents grade value)] × 100%
Control efficiency (%)=[(control group disease index-processing group disease index)/control group disease index] × 100%
According to test result it is found that the microorganism formulation containing bacillus subtilis strain of the invention is to banana chrysanthemum Euclidean
Squirter 4 times duplicate greenhouse pot culture control efficiency caused by bacillus are respectively 97.67%, 96.68%, 98.46%,
98.10%, average out to 97.73%.
Experimental data shows: the microorganism system of the anti-banana Erwinia chrysanthemi containing bacillus subtilis of the present invention
Agent also shows significant control efficiency in potting banana seedlings, puts down to squirter caused by banana Erwinia chrysanthemi
Equal control efficiency is 97.73%, all has preferable fungistatic effect in vitro and live test.Show withered grass of the invention
Bacillus and its microorganism formulation can have significant in vitro and living body preventive effect to Erwinia chrysanthemi, and prevention and treatment Erwinia chrysanthemi draws
The squirter significant effect risen.
Comparative example 1
The bacillus subtilis of the application is carried out in anti-konjak soft rot (cause of disease according to the method for embodiment 2 and embodiment 3
Bacterium is carrot soft rot Pectinatus (Pectobacteriumcarotovorum subsp.carotovorum), and number is15713) experiment.
Antibacterial Activity test and greenhouse pot culture live test are carried out respectively to the bacillus subtilis of the application.
By test, bacillus subtilis, without bacteriostatic activity, does not generate inhibition zone to konjak soft rot.Bacillus subtilis
4 duplicate greenhouse pot culture control efficiency to konjak soft rot are respectively 20.15%, 16.66%, 13.26% and
15.29%.
The preferred embodiment of the present invention has been described above in detail, still, during present invention is not limited to the embodiments described above
Detail within the scope of the technical concept of the present invention can be with various simple variants of the technical solution of the present invention are made, this
A little simple variants all belong to the scope of protection of the present invention.
It is further to note that specific technical features described in the above specific embodiments, in not lance
In the case where shield, can be combined in any appropriate way, in order to avoid unnecessary repetition, the present invention to it is various can
No further explanation will be given for the combination of energy.
In addition, various embodiments of the present invention can be combined randomly, as long as it is without prejudice to originally
The thought of invention, it should also be regarded as the disclosure of the present invention.
Claims (8)
1. a kind of bacillus subtilis (Bacillus subtilis), the deposit number of the bacillus subtilis is CGMCC
No.15506。
2. a kind of preparation method of microorganism formulation, includes the following steps:
(1) bacillus subtilis described in claim 1 carries out solid culture in solid medium and obtains test tube species；
(2) liquid seed culture medium is prepared, and is inoculated with test tube species progress Liquid Culture in the liquid seed culture medium and is made
Liquid seeds；
(3) liquid fermentation medium is prepared, and is inoculated with liquid seeds in the liquid fermentation medium and ferments；
The method also includes the fermentation liquids obtained to fermentation to purify；
The method of purification of fermentation liquid includes: that fermentation liquid is extracted using organic solvent, removes organic solvent later；The organic solvent
For acetone.
3. the preparation method of microorganism formulation according to claim 2, wherein in step (1), the bacillus subtilis
It is inoculated with by the way of inclined plane inoculating, the solid culture cultivates 40-50h under conditions of temperature is 25-30 DEG C；
The solid medium includes: glucose 10-12g/L, agar 15-20g/L, beef extract 3-5g/L, yeast extract 1-
3g/L, peptone 10-15g/L, pH=6.5-7.5.
4. the preparation method of microorganism formulation according to claim 2, wherein in step (2), test tube species are inoculated into liquid
In seed culture medium, the Liquid Culture cultivates 45- under conditions of temperature is 25-30 DEG C, revolving speed is 200-250r/min
50h；
The liquid seed culture medium includes: peptone 10-15g/L, beef extract 3-5g/L, sodium chloride 10-12g/L, pH=
6.5-7.5。
5. the preparation method of microorganism formulation according to claim 2, wherein in step (3), liquid seeds are sent out in liquid
In ferment culture medium according to 10-15 volume % inoculum concentration be inoculated with, it is described fermentation temperature be 25-30 DEG C, revolving speed 200-250r/
45-50h is handled under conditions of min；
The liquid fermentation medium includes: sucrose 20-23g/L, peptone 10-15g/L, yeast extract 5-8g/L, di(2-ethylhexyl)phosphate
Hydrogen potassium 3-5g/L, ammonium sulfate 5-8g/L, calcium carbonate 2-4g/L, pH=6.5-7.5.
6. the system of microorganism formulation described in any one of bacillus subtilis described in claim 1 or claim 2-5
Application of the microorganism formulation that Preparation Method is prepared in anti-squirter.
7. application according to claim 6, the pathogenic bacteria of the squirter are Erwinia chrysanthemi (Erwinia
chrysanthemi)。
8. the system of microorganism formulation described in any one of bacillus subtilis described in claim 1 or claim 2-5
The microorganism formulation that Preparation Method is prepared is inhibiting the application in Erwinia chrysanthemi.
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| CN101165171A (en) * | 2007-09-30 | 2008-04-23 | 华中农业大学 | Bacillus subtilis BSn5 with bacteriostasis active to carrot soft rot Erwinia and bacteriostasis protein APn5 |
| CN103451135A (en) * | 2013-09-09 | 2013-12-18 | 云南农业大学 | Bacillus subtilis M3 and application thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101165171A (en) * | 2007-09-30 | 2008-04-23 | 华中农业大学 | Bacillus subtilis BSn5 with bacteriostasis active to carrot soft rot Erwinia and bacteriostasis protein APn5 |
| CN103451135A (en) * | 2013-09-09 | 2013-12-18 | 云南农业大学 | Bacillus subtilis M3 and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| Bacillus subtilis BS 107 as an antagonist of potato blackleg and soft rot bacteria;B.M. Sharga and G.D. Lyon;《Can. J. Microbiol.》;19981231;第44卷;全文 * |
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